CN109975544A - A kind of PEDV IgY antibody colloidal gold test strip and its preparation method and application - Google Patents
A kind of PEDV IgY antibody colloidal gold test strip and its preparation method and application Download PDFInfo
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- CN109975544A CN109975544A CN201910342175.3A CN201910342175A CN109975544A CN 109975544 A CN109975544 A CN 109975544A CN 201910342175 A CN201910342175 A CN 201910342175A CN 109975544 A CN109975544 A CN 109975544A
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Abstract
The invention discloses a kind of PEDV IgY antibody colloidal gold test strips and its preparation method and application, sample pad, bonding pad, nitrocellulose filter and the water absorption pad that the test strips include PVC bottom plate and are successively overlapped on PVC bottom plate, wherein, the bonding pad is the colloidal gold pad for being coated with rabbit-anti chicken IgG- colloidal gold composite, and the nitrocellulose filter is equipped with by the coated nature controlling line of goat anti-rabbit igg and the coated detection line of PEDV recombinant antigen.Compared with prior art, the present invention has filled up blank of the PEDV IgY antibody at present without specific aim detection reagent, compares more conventional ELISA antibody detection method, and enhanced convenience of the present invention is quick.
Description
Technical field
The present invention relates to the rapid detection technical fields of PEDV IgY antibody (PEDV Yolk antibody), more particularly to one kind
PEDV IgY antibody colloidal gold test strip and its preparation method and application.
Background technique
Porcine Epidemic Diarrhea substance be Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus,
PEDV), it is single strand plus RNA virus, belongs to I class coronavirus, because it can be broadcast by excrement oral instructions, contact propagation and droplet transmission
Different sexes, the pig at age and kind are infected, are caused popular with the pig of the pathogenetic features such as acute water sample diarrhea, vomiting, dehydration
Diarrhea (porcine epidemic diarrhea, PED) becomes the main infection venereal disease original for currently endangering the production of world's pig industry
One of body, how prevention and control PEDV become Chinese scholar research major tasks.Currently, the vaccine for PEDV is less, and no matter
Import or domestic vaccine effect are bad, and PEDV Yolk antibody finds have preferably as feed addictive to PEDV in use
Therapeutic effect and prevention effect.But in the market temporarily without the reagent for being directed to the detection of PEDV Yolk antibody.
Colloidal gold immunochromatographimethod technology has obtained increasingly wide as a kind of new immunological method in biomedical each field
General application.It is after traditional three big immunolabelling techniques it is another it is more mature, be used widely it is immune labeled
Technology.After sample to be tested is added on sample film, due to the capillarity of miillpore filter, make antigen-antibody reaction in solid phase
It is quickly carried out on film, as long as detecting general 5~10min will go out as a result, significantly compared to other methods (such as ELISA needs 1~2h)
Shorten detection time.Its test result brings judgement with macroscopic colour developing item, does not need special instrument and equipment, only needs
Test strips or diafiltration kit are wanted, as long as sample does very simple processing or do not need to do pre-treatment.And have cost is relatively low,
Easy to operate, stable reagent is not influenced by extraneous factors such as temperature, is convenient and efficient, special sensitivity, stability is strong, result judgement is straight
The advantages that sight, thus it is particularly suitable for quickly checking that there is huge development potentiality and wide application prospect on site, it represents
Diagnostic reagent is simple and quick, convenient for universal developing direction.The present invention is prepared for the indirect method colloidal gold inspection of PEDV IgY a kind of
Test paper slip.
Summary of the invention
In order to solve problem above, the present invention proposes a kind of PEDV IgY antibody colloidal gold test strip and its preparation side
Method and application, to provide a kind of method quickly detected for PEDV Yolk antibody.
The present invention is realized using following technical scheme:
The present invention provides a kind of PEDV IgY antibody colloidal gold test strip, including PVC bottom plate and successively it is overlapped on
Sample pad, bonding pad, nitrocellulose filter and water absorption pad on PVC bottom plate, wherein the bonding pad is to be coated with rabbit-anti chicken
The colloidal gold pad of IgG- colloidal gold composite, the nitrocellulose filter be equipped with by the coated nature controlling line of goat anti-rabbit igg and
The coated detection line of PEDV recombinant antigen.
The present invention also provides the preparation methods of above-mentioned PEDV IgY antibody colloidal gold test strip, including following step
It is rapid:
(1) the PEDV recombinant antigen of preparation purifying；
(2) nitrocellulose filter is prepared: after the PEDV recombinant antigen of goat anti-rabbit igg and purifying is diluted respectively, coating
On the nature controlling line and detection line of nitrocellulose filter；
(3) colloidal gold is prepared；
(4) colloid gold label rabbit anti-chicken IgG: taking rabbit anti-chicken IgG, and colloidal gold is mixed with rabbit anti-chicken IgG, and PBS is used in centrifugation
Precipitating is resuspended in solution, obtains rabbit-anti chicken IgG- colloidal gold composite；
(5) preparation gold mark release pad: rabbit-anti chicken IgG- colloidal gold composite is uniformly sprayed on bonding pad, sufficiently dry,
Obtain colloidal gold pad；
(6) assembling of test strips: in dry environments, by sample pad, colloidal gold pad, the nitrocellulose filter being coated with and
Water absorption pad is pasted onto PVC board, is cut, and test strips are obtained.
It is further preferred that the step of PEDV recombinant antigen of preparation purifying includes: according to known PEDV N protein base
Because sequence prepares the Escherichia coli recombinant strain of PEDV N protein, by recombinant bacterium fermentation expression and it is centrifuged acquisition thallus, thallus is broken
Broken, supernatant is crossed affinity chromatographic column by centrifuging and taking supernatant, the PEDV recombinant antigen as purified.
It is further preferred that in the step of preparing nitrocellulose filter, the PEDV recombinant antigen of goat anti-rabbit igg and purifying
Final concentration of 1mg/ml is diluted to the PBS of 100mM respectively, then is sprayed with the discharge rate of 0.8 μ L/cm and is coated on nature controlling line and detection
It is sufficiently dry on line.
It is further preferred that the method for preparing colloidal gold is trisodium citrate reduction method, step includes: to take the mass fraction to be
The HAuCl of 0.01%-0.02%4Aqueous solution 100ml, heating are boiled, and the citric acid three that mass fraction is 1%-5% is rapidly added
Sodium water solution 1ml continues to boil, and solution becomes grey by faint yellow, then changes into black, then gradually stable at claret,
Colloidal gold solution is made.
It is further preferred that the step of colloid gold label rabbit anti-chicken IgG includes: that high-purity rabbit anti-chicken IgG is taken to be dissolved in 0.1-
In the PBS of 0.2ml, the pH value of colloidal gold solution is adjusted to 6.9；Rabbit anti-chicken IgG is mixed with colloidal gold by following combination: every
10ml colloidal gold solution is added in 60mg rabbit anti-chicken IgG；After mixing 3min, the PEG6000 room temperature that mass fraction is 0.05% is added
After effect combines 15min, first 2000r/min low-speed centrifugal 10min, then 12000r/min high speed centrifugation 30min；Use 100mM/L
The PBS solution containing 0.2% mass fraction Proclin300 of pH7.6 precipitating is resuspended, as rabbit-anti chicken IgG- colloidal gold is compound
Object.
It is further preferred that discharge rate of the rabbit-anti chicken IgG- colloidal gold composite on glass fibre element film is 10 μ L/cm.
It is further preferred that the method for the assembling of test strips are as follows: be ready in dry environments sample pad, colloidal gold pad,
Coated nitrocellulose filter, water absorption pad and PVC bottom plate stick the nitrocellulose filter being coated with, nitric acid in PVC bottom plate center
Cellulose membrane upper limb pastes water absorption pad, and nitrocellulose filter lower edge pastes colloidal gold pad, and colloidal gold pad lower edge pastes sample pad, complete
Test paper plate is cut into strip after.
The present invention also provides above-mentioned PEDV IgY antibody colloidal gold test strips in detection PEDV IgY antibody
Using step includes: that will test test paper to lie against on desktop, takes test serum sample 50-100 μ L, and sample pad is added, and room temperature is quiet
After setting 15min, result is determined according to following situations:
Test paper is effective: purplish red colo(u)r streak occurs in nature controlling line, that is, illustrates that this test paper is effective；
It is negative: only purplish red colo(u)r streak occur in nature controlling line, colo(u)r streak do not occur in detection line；
It is positive: a purplish red colo(u)r streak occur in nature controlling line and detection line；
And the colo(u)r streak depth is directly proportional to antibody titer, i.e., antibody titer is higher, and colo(u)r streak is deeper.
The beneficial effects of the present invention are: the present invention provides a kind of PEDV IgY antibody colloidal gold test strip and its systems
Preparation Method and application, after being detected using Test paper, positive findings illustrate detected sample PEDV IgY antibody positive and reach
Desired antibody level, the antibody level that negative findings illustrate sample PEDV IgY negative antibody or not up to require, the examination
Paper slip can detect rapidly PEDV IgY antibody level, fill up blank of the PEDV IgY antibody at present without specific aim detection reagent,
Compared to more conventional ELISA antibody detection method, enhanced convenience of the present invention is quick.
Detailed description of the invention
Fig. 1 is PEDV IgY antibody immune colloidal gold detection test paper strip structural schematic diagram；Wherein, A is test strips internal junction
Structure schematic diagram, B are schematic diagram of product structure after assembling；
Fig. 2 is PEDV IgY antibody immune colloidal gold detection test paper strip testing result schematic diagram；Wherein, A is pattern detection
Result figure；B is testing result schematic diagram.
Specific embodiment
In order to be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, tie below
Conjunction is specifically illustrating, and the present invention is further explained.
Embodiment 1
1, material
The present embodiment method therefor is known to those skilled in the art the conventional method of dawn unless otherwise instructed, used
The materials such as reagent be commercially available products unless otherwise instructed.
2, method
The preparation of the PEDV recombinant antigen of 2.1 purifying
PEDV N protein gene order, the Escherichia coli for sending Huada gene company to prepare PEDVN albumen are obtained from GENBANK
Recombinant bacterium by recombinant bacterium fermentation expression and is centrifuged acquisition thallus, and thallus using high-pressure homogeneous crusher machine and is used ultrasonication
Supernatant is crossed affinity chromatographic column by instrument second-time breakage, centrifuging and taking supernatant, the PEDV recombinant antigen as purified.
2.2 prepare nitrocellulose filter 4
Goat anti-rabbit igg and the PEDV recombinant antigen of purifying are diluted to final concentration of 1mg/ respectively with the PBS of 100mM
Ml, the nature controlling line C and detection line T being then uniformly sprayed on respectively on nitrocellulose filter 4, discharge rate are 0.8 μ L/cm, sufficiently dry
It is dry.
The preparation of 2.3 colloidal golds
Using trisodium citrate reduction method, 10-40nm colloidal gold is prepared, the specific operation method is as follows: taking the mass fraction to be
The HAuCl of 0.01%-0.02%4Aqueous solution 100ml, heating are boiled, and the citric acid three that mass fraction is 1%-5% is rapidly added
Sodium water solution 1ml continues to boil about 5min, and partial size 10-40nm gold particle is made, and flaxen gold chloride water can be observed at this time
Solution quickly becomes grey after trisodium citrate addition, continues and changes into black, then gradually stable at claret, is cooled to room
It is kept in dark place for 4 DEG C after temperature.
2.3 colloid gold label rabbit anti-chicken IgGs
It takes high-purity rabbit anti-chicken IgG to be dissolved in the PBS of 0.1-0.2ml, the pH value of colloidal gold solution is adjusted to 6.9；By with
Lower combination mixes rabbit anti-chicken IgG with colloidal gold: the 40nm colloidal gold solution of 10ml is added in every 60mg rabbit anti-chicken IgG；Mixing
After 3min, after the PEG6000 room temperature effect that addition mass fraction is 0.05% combines 15min, first low-speed centrifugal, 2000r/min,
10min, then high speed centrifugation, 12000r/min, 30min.It is resuspended with the PBS (containing 0.2%Proclin300) of 100mM/L pH7.6
Precipitating, as rabbit-anti chicken IgG- colloidal gold composite.
2.4 preparation gold mark release pads
The rabbit-anti chicken IgG- colloidal gold composite of above-mentioned preparation is uniformly sprayed on glass fibre element according to the discharge rate of 10 μ L/cm
It is sufficiently dry on film, obtain colloidal gold pad 3.
The assembling of 2.5 test strips
It gets out water absorption pad 5, sample pad 2, PVC bottom plate 1 in drying room, sticks step 2.2 packet in 1 center of PVC bottom plate
By good nitrocellulose filter 4,4 upper limb of nitrocellulose filter pastes water absorption pad 5,4 lower edge gluing steps 2.4 of nitrocellulose filter
The colloidal gold pad 3 of preparation, 3 lower edge of colloidal gold pad paste sample pad 2, and it is 1-2mm that each component, which is overlapped mutually part, after the completion with sanction
The test strips that the test paper plate posted is cut into 4.1mm wide by machine are cut, as shown in Figure 1A.The test strips cut are fitted into getting stuck, then
Test strips and desiccant are sealed in aluminium foil bag, complete the assembling of product, as shown in Figure 1B.
Test paper use provided by the present invention can be followed the steps below with result judgement method: test strips are put down
It is put on desktop, takes test serum sample 50-100 μ L, sample pad 2 is added, after being stored at room temperature 15min, as shown in Fig. 2, according to
Following situations determines result:
Test paper is effective: purplish red colo(u)r streak occurs in nature controlling line C, that is, illustrates that this test paper is effective；
It is negative: only purplish red colo(u)r streak occur in nature controlling line C, colo(u)r streak do not occur in detection line T；
It is positive: a purplish red colo(u)r streak occur in nature controlling line C and detection line T；
And the colo(u)r streak depth is directly proportional to antibody titer, i.e., antibody titer is higher, and colo(u)r streak is deeper.
3, test strips detection effect is verified
The antibody level required when being dispatched from the factory according to PEDV IgY antibody, takes PEDV IgY antibody, with the PBS solution of 100mM
100ng/ml concentration, 200ng/ml concentration, 400ng/ml concentration, 800ng/ml concentration is diluted to set simultaneously as test sample
Blank control is set, test strips and enzyme linked immunosorbent assay (ELISA) detection antibody concentration of the invention are utilized respectively, it is as a result as follows
Shown in table 1:
Table 1: distinct methods detect PEDV IgY antibody concentration
Table 1 is as can be seen that the testing result of colloidal gold strip of the invention has height consistent with ELISA testing result
Property, illustrate that the accuracy rate of colloidal gold strip of the present invention is 100%, detects PEDV IgY using the colloidal gold strip of the application
Antibody and its content are feasible.Since the colo(u)r streak depth is directly proportional to antibody titer, antibody titer is higher, and colo(u)r streak is deeper, can be by
This detects whether commercially available PEDV IgY antibody product has reached factory antibody level, to judge whether the product of purchase is false
Goods is adulterated.
The basic principles, main features and advantages of the invention have been shown and described above.The technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, and the above embodiments and description only illustrate the principle of the present invention,
Without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes and improvements are all fallen
Enter in scope of the claimed invention.The scope of the present invention is defined by the appended claims and its equivalents.
Claims (9)
1. a kind of PEDV IgY antibody colloidal gold test strip, the sample that including PVC bottom plate and is successively overlapped on PVC bottom plate
Product pad, bonding pad, nitrocellulose filter and water absorption pad, which is characterized in that the bonding pad is to be coated with rabbit-anti chicken IgG- colloid
The colloidal gold pad of Au composite, the nitrocellulose filter is equipped with to be recombinated by the coated nature controlling line of goat anti-rabbit igg and PEDV
The detection line of antigen coat.
2. a kind of preparation method of PEDV IgY antibody colloidal gold test strip as described in claim 1, which is characterized in that
The following steps are included:
(1) the PEDV recombinant antigen of preparation purifying；
(2) it prepares nitrocellulose filter: after the PEDV recombinant antigen of goat anti-rabbit igg and purifying is diluted respectively, being coated on nitre
On nature controlling line and detection line on acid cellulose film；
(3) colloidal gold is prepared；
(4) colloid gold label rabbit anti-chicken IgG: taking rabbit anti-chicken IgG, and colloidal gold is mixed with rabbit anti-chicken IgG, and PBS solution is used in centrifugation
Precipitating is resuspended, obtains rabbit-anti chicken IgG- colloidal gold composite；
(5) preparation gold mark release pad: rabbit-anti chicken IgG- colloidal gold composite is uniformly sprayed on bonding pad, sufficiently dry, is obtained
Colloidal gold pad；
(6) assembling of test strips: in dry environments, by sample pad, colloidal gold pad, the nitrocellulose filter being coated with and water suction
Pad is pasted onto PVC board, is cut, and test strips are obtained.
3. a kind of preparation method of PEDV IgY antibody colloidal gold test strip according to claim 2, feature exist
In the step of preparing the PEDV recombinant antigen of purifying includes: to prepare PEDV N egg according to known PEDV N protein gene order
White Escherichia coli recombinant strain by recombinant bacterium fermentation expression and is centrifuged acquisition thallus, and by bacterial cell disruption, centrifuging and taking supernatant will be upper
It is clear to cross affinity chromatographic column, the PEDV recombinant antigen as purified.
4. a kind of preparation method of PEDV IgY antibody colloidal gold test strip according to claim 2, feature exist
In in the step of preparing nitrocellulose filter, the PEDV recombinant antigen of goat anti-rabbit igg and purifying is dilute with the PBS of 100mM respectively
It releases to final concentration of 1mg/ml, then is sprayed on nature controlling line and detection line with the discharge rate of 0.8 μ L/cm, it is sufficiently dry.
5. a kind of preparation method of PEDV IgY antibody colloidal gold test strip according to claim 2, feature exist
In the method for preparing colloidal gold is trisodium citrate reduction method, and step includes: to take mass fraction for 0.01%-0.02%
HAuCl4Aqueous solution 100ml, heating are boiled, and the trisodium citrate aqueous solution 1ml that mass fraction is 1%-5% is rapidly added, after
Continuous to boil, solution becomes grey by faint yellow, then changes into black, then gradually stable at claret, and gold particle is made.
6. a kind of preparation method of PEDV IgY antibody colloidal gold test strip according to claim 2, feature exist
It include: that high-purity rabbit anti-chicken IgG is taken to be dissolved in the PBS of 0.1-0.2ml in the step of, colloid gold label rabbit anti-chicken IgG, by colloid
The pH value of gold solution is adjusted to 6.9；Mix rabbit anti-chicken IgG with colloidal gold by following combination: every 60mg rabbit anti-chicken IgG is added
10ml colloidal gold solution；After mixing 3min, after the PEG6000 room temperature effect that addition mass fraction is 0.05% combines 15min, first
2000r/min low-speed centrifugal 10min, then 12000r/min high speed centrifugation 30min；Contain 0.2% with the pH7.6 of 100mM/L
Precipitating, as rabbit-anti chicken IgG- colloidal gold composite is resuspended in the PBS solution of mass fraction Proclin300.
7. a kind of preparation method of PEDV IgY antibody colloidal gold test strip according to claim 2, feature exist
In discharge rate of the rabbit-anti chicken IgG- colloidal gold composite on bonding pad is 10 μ L/cm.
8. a kind of preparation method of PEDV IgY antibody colloidal gold test strip according to claim 2, feature exist
In the method for the assembling of test strips are as follows: be ready in dry environments sample pad, colloidal gold pad, coated nitrocellulose filter,
Water absorption pad and PVC bottom plate stick the nitrocellulose filter being coated in PVC bottom plate center, and nitrocellulose filter upper limb, which is pasted, to be inhaled
Water cushion, nitrocellulose filter lower edge paste colloidal gold pad, and colloidal gold pad lower edge pastes sample pad, test paper plate is cut into inch strips after the completion
Shape.
9. a kind of PEDV IgY antibody colloidal gold test strip as described in claim 1 is in detection PEDV IgY antibody
Using, which is characterized in that it will test test paper and lie against on desktop, take test serum sample 50-100 μ L, sample pad, room temperature is added
After standing 15min, result is determined according to following situations:
Test paper is effective: purplish red colo(u)r streak occurs in nature controlling line, that is, illustrates that this test paper is effective；
It is negative: only purplish red colo(u)r streak occur in nature controlling line, colo(u)r streak do not occur in detection line；
It is positive: a purplish red colo(u)r streak occur in nature controlling line and detection line；
And the colo(u)r streak depth is directly proportional to antibody titer, i.e., antibody titer is higher, and colo(u)r streak is deeper.
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| CN111551744A (en) * | 2020-05-15 | 2020-08-18 | 安徽中起生物科技有限公司 | Newcastle disease virus N protein IgY antibody colloidal carbon detection test paper and application thereof |
| CN111551745A (en) * | 2020-05-15 | 2020-08-18 | 安徽中起生物科技有限公司 | Colloidal gold test paper and method for detecting avian influenza virus H7N9 subtype N protein IgY antibody |
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Inventor after: Bian Chuanzhong Inventor after: Dong Shijian Inventor after: Pan Xiang Inventor after: Liu Jiarong Inventor before: Bian Chuanzhong Inventor before: Dong Shijian Inventor before: Pan Xiang Inventor before: Liu Jiarong |
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